• 한국미생물·생명공학회지
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• 제49권1호
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• pp.45-56
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• 2021

Improved amylases were developed from protoplast fusants of two amylase-producing Aspergillus species. Twenty regenerated fusants were screened for amylase production using Remazol Brilliant Blue agar. Crude enzyme extracts produced by solid state fermentation of rice bran were assayed for activity. Three variable factors (temperature, pH and enzyme type) were optimized to increase the amylase activity of the parents and selected fusants using rice bran medium and solid state fermentation. Analysis of this optimization was completed using the Central Composite Design (CCD) of the Response Surface Methodology (RSM). Amylase activity assays conducted at room temperature and 80℃ demonstrated that Aspergillus designates, T5 (920.21 U/ml, 966.67 U/ml), T13 (430 U/ml, 1011.11 U/ml) and T14 (500.63 U/ml, 1012.00 U/ml) all exhibited improved function making them the preferred fusants. Amylases produced from these fusants were observed to be active over the entire pH range evaluated in this study. Fusants T5 and T14 demonstrated optimal activity under acidic and alkaline conditions, respectively. Fusants T13 and T14 produced the most amylase at 72 h while parents TA, TC and fusant T5 produced the most amylase after 96 h of incubation. Response surface methodology examinations revealed that the enzyme from fusant T5 was the optimal enzyme demonstrating the highest activity (1055.17 U/ml) at pH 4 and a temperature of 40℃. This enzyme lost activity with further increases in temperature. Starch hydrolysis using fusant T5 gave the highest yield of glucose (1.6158 g/100 ml). The significant activities of the selected fusants at 28 ± 2℃ and 80℃ and the higher sugar yields from cassava starch hydrolysis over their parental strains indicate that it is possible to improve amylase activity using the protoplast fusion technique.

• 동아시아식생활학회지
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• 제5권1호
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• pp.93-99
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• 1995

For the improvement of the L-lysine productivity of Brevibacterium flavum and Corynebacterium glutamicum, fusants were induced by interspecific protoplast fusion of Bacillus subtilis with C. glutamicum and B. flavum. The following results were obtained through protoplast formation of strains condition of protoplast fusion, characteristics of the fusants, and the productivity of lysine form starch. B. flavum BF-5 and C. glutamicum protoplasts were made by the treatment of 0.3unit/$m\ell$ of penicillin G at the early stationary growth phase for 2 hours followed by incubation with 10mg/$m\ell$ of lysozyme at 37$^{\circ}C$ for 120 min. When a mixture of the protoplast was treated with 30% PEG(M.W.6,000) solution containing 50mM CaCl2 at optimal conditions, the intergeneric fusion frequency between protoplasts of C. glutamicum CG-2 and B. subtilis BD 224 was 7.1${\times}$105. The genetic properties on the L-lysine producing fusants were compared with those of parental strains. As a results, the intergeneric fusants were completed in each auxotrophic requirement, resistances for S-(2-amino-ethyl)-L-cysteine and kanamycine were confirmed. And one of fusants selected, FBB-41 were found to be genetically stable fusants. The aspartokinase activity of FBB-41 strain increased than that of the parent strain.

• 미생물학회지
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• 제28권4호
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• pp.311-317
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• 1990

Intraspecific and interspecific protoplast fusions in/between C. thermocellum and C. thermohydrosulfuricum were studied. Protoplast fusions were well induced in 30-40% PEG solution, however, their fusion frequencies were low level of 1.2*10$^{-7}$ for intraspecific fusion of C. thermocellum, $6.7*10^{-7}$ for C. thermohydrosulfuricum, and 4.2*10$^{-7}$ for interspecific fusion between above two Clostridia, respectively. Most fusants were unstable and segregated after 3 subcultures. Relatively stable intraspecific C. thermocellum fusant FTT17, intraspecific C. thermohydrosulfurecum fusant FSS22 and interspecific fusant FTS3, which were stable after several subcultures, were selected and properties of fusants were further investigated, Phenotypes of the fusants were similar with wild types mostly in cellular morphology, carbon source assimilation and enzyme activities. However they were differed in assimilation of pyruvic acid and sorbitol as carbon source. The DNA contents of fusants were slightly increased compared with wild types. Ethanol production by intraspecific and/or interspecific fusants was not increased, however, acetic acid production as byproduct was decreased or not detected, which indicates that industrial thermophilic anaerobes can be improved by means of protoplast fusion of two strains.

• Journal of Microbiology and Biotechnology
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• 제7권1호
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• pp.25-31
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• 1997

Protoplast fusion between L. casei KCTC 1121 and L. acidophilus 88 was attempted to obtain improved strains. The fusants produced a bacteriocin against indicator strains, making a smaller inhibition zone compared to that of L. acidophilus 88. After culturing for 2 months on selective medium, the selected fusants were still stable without segregation. Fusants showed higher lipase activity compared to those of the two parent strains. Fusant No.4, 11, and 15 exhibited excellent lactic acid productivity. Fusant No.4 and 15 exhibited improved proteolysis ability compared to the two parent strains. Whereas L. casei possessed both ${\beta}-galactosidase$ and $phospho-{\beta}-galactosidase$ activities, and L. acidophilus 88 had only ${\beta}-galactosidase$ activity, the fusants had both the intermediate enzyme activities. Cell size of the fusants was greater than that of the parents.

• 미생물학회지
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• 제28권1호
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• pp.55-64
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• 1990

원형질체 융합을 통하여 killer 효모의 유전형질을 기존의 ethanol 발효효묘에 도입하므로서 야생의 killer 효모에 저항성을 가지고, 오염효모를 치사시킬 수 있으며, ethanol 발효능도 유지하는 새로운 효모균주를 개발하였다. 먼저 융합주의 생리적인 특성을 검토한 바, 융합주는 양천주에 비하여 세 적이 크고, DNA 함량이 높았으며, 증식도는 친주와 유사한 경향이었다. 또 융합주를 최소배지에서 보존하는 것이 안정성을 높이는 방법이었고, 7일 간격으로 7회 계대배양하므로서 유전적으로 안정화시킨 융합주를 6개월 간 계속 사용한 결과 segregant가 전혀 나타나지 않았으므로 매우 안정하였다. 또한 융합주는 핵 및 포자를 형성함을 관찰 할 수 있었고, TTC 정색반응에서 적색 및 pink 색을 띄었으며, 탄소원의 자화성 및 발효능은 천주와 유사하였는데, KCI, NaCl, sodium propionate alc cycloheximide 등에 대한 내성도 양친주의 한쪽을 따랐다. 그리고, 융합주를 20% glucose와 sucrose에서 72시간 및 60시간 배양했을 때, FWKS 260의 경우 각각 9.6v/v% 및 9.8v/v%의 ethanol을 생성하였고, 감수성주와 혼합배양한 결과 FWKS260의 경우 48시간 이후에는 감수성주를 거의 발견할 수 없었으며, 전주 및 융합주의 dsRNA plasmid를 추출하여 전기 2.5kb의 L 및 M dsRNA plasmidsms는 서로 연관성이 있으며, killer toxin 분비 및 저항성을 나타내는 유전자를 지배하는 것은 M dsRNA plasmid임을 확인할 수 있었다. 수 있었다.

• 미생물학회지
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• 제28권1호
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• pp.47-54
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• 1990

섬유소를 분해할 수 있는 Cellutomonas flavigena NCIB 12901 과 Cellulomonas bibula NCIB 8142의 종간 원형질체 융합을 하고 그로부터 얻은 융합체의 특성을 조사하였다. C. bibuta의 원형질체는 lysozyme(600Ieιllulomoηas libula NCIB 8142의 종간 원형 섣처l 융 한응 하고:1로부터 얻은융합셰익 특성을 조사하였다 . C bibula의 원형선셰는 lysozyme $600{\mu}{\textrm{g}}$/ml)를 6시간 처리하여 얻었으며, 삼투안정제로는 O.5M sorbitol의 최저이었고 원형질체 재생률은 6.7%였다. 원형질체 융합은 25mM $CaCl_{2}$를 포함하는 40% PEG (M.W.6000)를 $30^{\circ}C$에서 30분간 처리함으로 수행하였고 C. bibuιl와 C. flavigeηa의 융합 빈도는 $5\times 10^{-4}$으로 원형질체 형성 및 합체를 얻었고, 모균주와 융합체의 영양요구성, 항생물질 내성, 효소활성, 세포벽 성분, 운동성 등을 비교하여 융합체는 모균주의 재조합체임을 밝히었다.

• 한국미생물·생명공학회지
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• 제16권3호
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• pp.182-186
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• 1988

원형질체 융합기술에 의한 Penicillium verruculosum 균주개량 가능성을 검토하였다. 영양요 구변이주간의 원형질 융합체 165주중 cellulase 고생산 융합체로 선별된 6주의 cellulase 활성은 야생주에 비해 2배이상, 모균주에 비해 1.2-4.4배의 증가를 나타냈다. Cellulase 고생산 융합체는 유전적 안정성조사, DNA 함량 및 핵염색 비교검토를 통해 핵융합이 일어난 것으로 확인되었고, 핵형이 이수체이며 4 개월 이상의 계대배양 후에도 자연형질 분리가 거의 일어나지 않는 것으로 보아 이들 융합체의 안정성이 매우 높은 것으로 인정되었다.

• 한국미생물·생명공학회지
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• 제14권5호
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• pp.365-369
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• 1986

새로운 alcohol발효성 효모 균주 개발을 목적으로 S. diastaticus 와 C. tropicalis간의 세포 융합을 하여 얻은 fusant의 성질에 대해서는 제3보$^{(10)}$에서 발표한 바이다. 본 보에서 는 fusant의 glucoamylase와 pullulanase activity와 glucoamylase의 성질 및 발효력을 조사하였다. glucoamylase activity는 parent보다 fusant가 1.5배 높은 활성을 나타내었고 pullulanase activity 역시 두배의 높은 활성을 나타내었다. glucoamylase의 성질을 조사한 바 온도, pH에서 비슷한 경향을 나타내었으며 발효력을 조사하기 위하여 정치법에서는 기질을 soluble starch로 한 것 보다 liquefied potato starch에서 alcohol 생성력이 2배 증가되었으며 발효력 또한 더 나았다. 15% 액화한 potato starch를 기질로하여 jar-fermenter에서 발효시켰을 때 당화율 94%에서 생성된 alcohol이 7.8% (v/v)이고 소비된 당에 대한 발효율은 78%였다.

• 한국균학회지
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• 제17권4호
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• pp.197-201
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• 1989

야생형(野生形)인 만가닥버섯 Lyophyllum ulmarium(=Hypsizigus marmoreus) ASI 8007에서 분리한 분열자(分裂子)와 잔나비걸상버섯 영양요구주 Ganoderma applanatum ASI 7-18 (cys met)의 원형질체(原形質體)를 polyethylene glycol로 융합(融合)하여 융합주를 얻었다. 융합주는 GCM(Ganoderma complete modium)에서 양친균총이 혼합된 형태였으나 3회 계대 배양되면서 만가닥버섯 균총으로 변하여 균사에는 클램프연결체와 분열자를 지녔고, 만가닥과 거의 유사한 자실체를 형성하였다. 균사체로 esterase 동위효소(同位酵素)를 분석하였는데 만가닥이 1개 밴드, 잔나비걸상이 2개의 밴드를 가진데 비하여 융합주는 양친의 3개와 새로운 2개의 밴드를 나타내었다. 이들 융합주 상호간에는 균총형태, 생장속도, esterase 동위효소, 자실체 특성에 있어서 거의 차이가 없어 아주 안정되게 양친의 유전물질을 보유하였다.

• 한국식품영양과학회지
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• 제26권6호
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• pp.1237-1245
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• 1997

Interspecific fusants were made from the cells of two strains of Lactobacillus genus, a streptomycin resistant Lactobacillus sp. JC-7 and a kanamycin resistant L. acidophilus 88. The morphological and physiological properties of the fusants were examined by determining bacteriocin productivity, acid-producing activity, ability of carbohydrates utilization and three important enzyme activities. The fusants produced a bacteriocin against indicator strains and fusant No. 1, 4 exhibited a larger inhibition zone compared to that of L. acidophilus 88. $\beta$-Galactosidase, phospho-$\beta$-galactosidase, lipase activities and resistance to NaCl of Lactobacillus sp. JC-7 were better than those of L. acidophilus 88. Fusant No. 3 and 7 exhibited excellent lipase activities. Protease activity and acid productivity of L. acidophilus 88 were better than those of Lactobacillus sp. JC-7. Proteasse activities of all fusants were higher than those of parental strains, and expecially fusant No. 5 and 7 exhibited excellent proteolysis ability.